Control of microflora

ABSTRACT

The present invention relates to a method of controlling microorganisms adhering to the surfaces of biological material, by the administration of substances which alter the water-structure binding capacity of the surfaces and phase-boundaries. 
     Organic and inorganic salts, erythrosine or anti-oxidants are used to remove micro-organisms from the surfaces. Cucumine or derivatives thereof are used as adhesion-stimulating substances. 
     The invention also relates to preparations for performing the method, such as ointments, creams and bathing liquids containing the above-mentioned substances, as well as to dentifrices containing one or more organic or inorganic salts.

The present invention relates to a method of removing undesiredmicro-organisms, including those of pathogenic nature, attached tosurfaces, phase boundaries and interfaces. The invention also relates toa method of effecting an adhesion-stimulating result for desiredmicro-organisms by administering adhesion-stimulating substances. Theinvention also relates to preparations for achieving the abovementionedresults.

In the following the invention will be described first as applied to thecare of livestock, other applications being dealt with afterwards.

The present invention can be used to prevent the establishment ofpathogenic bacteria flora and to eliminate such flora alreadyestablished in pigs, calves and poultry. By "poultry" is meant chickens,turkeys, geese and ducks, for instance.

The mortality rate of such animals is at present high as a result of theestablishment and colonization of pathogenic bacteria in the stomach andintestines. Pathogenic bacteria oust the normal bacteria flora, adhereto the bonding seats on the walls of the intestines, give rise todisease symptoms such as diarrhoea and result in increased mortality.Some farms lose large numbers of animals following an outbreak ofpathogenic bacteria such as Escherichia coli, Salmonella typhimurium,Salmonella Sp., Shigella sp. and Clostridium perfringens.

Current methods of reducing the risk of outbreaks of the type mentionedabove can be divided into two categories:

1. Treatment with antibiotics

2. Oral administration of non-pathogenic bacteria in pure or mixedcultures

There are a number of drawbacks in treating animals with antibiotics,which makes the method less attractive.

Examples are:

Non-selectivity, which also causes a reduction in the natural intestinalflora;

High cost;

The appearance of resistant strains;

It is time-consuming and impractical when the farm worker cannot beresponsible for all steps of the treatment himself;

Legislative control of treatment with antibiotics.

It has been impossible to follow up successful laboratory experimentswith oral administration of non-pathogenic bacteria resulting in reducedrisk of diarrhoea in pigs, under full-scale conditions. Careful testswith commercial products containing, e.g. Lactobacillus acidophilus andLactobacillus bulgaricus have not given significant positive results.Neither has it been possible to colonize the intestines of pigs withStreptococcus faecium. It has not been possible to establishLactobacillus acidophilus in chicken intestines and there have been nosuccessful large-scale experiments with oral administration to chickens.

More recent experiments with plasmid-carrying strains to increaseadhesion appear to be futile due to instability of the plasmids anddifficulties in maintaining suitable selection pressure for theirpreservation.

Initial studies of the adhesion of bacteria to the intestinal wallsrevealed surprisingly that oral administration is anyway an extremelysuccessful method of preventing the establishment, colonization andgrowth of pathogenic bacteria in the stomach and intestines of pigs,calves and poultry.

The following points comprise the basis of the process forming thegrounds of the present invention is based:

1. The stomach and intestines is rendered substantially bacteria-free bytreating with anti-microbial substances or substances which adhere tothe walls and block the adhesion of bacteria.

2. Substances are used which increase the degree of adhesion of theorally administered non-pathogenic bacteria.

3. Non-pathogenic enterobacteria are cultivated in a special way toproduce specific protein-protrusions, fimbria, giving rise to greatlyincreased adhesion of bacteria to the bacteria surface of intestinalwalls.

Substances which may be used to remove bacteria from surfaces includeerythrosine, anti-oxidants, and organic or inorganic salts. Among thesalts which can be used are magnesium, aluminum, ammonium, beryllium,lithium and calcium salts. The various bacteria removing substances maybe used alone or in combination.

Substances which may be used to increase the degree of adhesion of thenon-pathegenic bacteria include curcumin and its derivatives. Curcuminalso known as1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione is thecoloring matter of tumeric, the rhizome of curcuma longa. Curcumin hasthe following structural formula: ##STR1##

The derivatives of curcumin which may be used as adhesion promotingsubstances include those of the following structural formula: ##STR2##Where N=1,2,3 . . . , X=1,2,3,4 . . . , and R₁ -R₅ may be same ordifferent, and may be for example, H, OH, OCH₃, OCH₂ CH₃, CH₃, CH₂ CH₃,COOH, or CH₂ COOH.

The curcumin may be administered in its pure form, or may beadministered in the form of oleo-resins and/or cleaned fractions of aplant extract containing curcumin, which also includes turmeric acid.

The invention is further illustrated with reference to the followingnon-limiting examples of its application:

EXAMPLE 1 RELATING TO PIGS AND CALVES

A. Erythrosine, anti-oxidants or MgSO₄ were mixed into the fodder ordrinking water in concentrations expressed in g/kg bodyweight and day0.06, 1, 0.5, respectively. Observation of laboratory animals (mice)with a scanning transmission electron microscope has revealed that thebacterial flora in the intestines were greatly reduced. Largebacteria-free areas were observed using erythrosine and anti-oxidantsand the use of MgSO₄ resulted in completely bacteria-free intestines.

B. Faeces samples from uncontaminated animals were coated with MacConkeyplates. The isolated samples were tested using a screening test, forEnterobacteriaceae in order to avoid Escherichia coli and Salmonellatypes. A suitable culture was selected and cultivated to produce fimbriawhich were checked with the yeast agglutination method. Intestinal wallwas at the same time taken with caecum and large intestine ofuncontaminated animals. Irreversibly bound bacteria plus intestinal wallwas homogenized and frimbria-carrying bacteria were produced asdescribed above.

Binding studies revealed that curcumine and similar compounds increasedthe degree of adhesion of Lactobacillus acidophilus to pig intestinesfive times.

The following bacteria preparations were produced for oral adminstrationto pigs in skimmed milk fermented with Lactobacillus acidophilus.Capsules (sterilized) were used containing 10¹⁰ /ml Streptococcuscaecalis or S. faecium, fimbriated Enterobacteria and curcumine oranalogous compounds, in sizes suited to the size of the animals.Curcumine is active if administered in amounts of 0.8 g/body-weight andday. This bacteria preparation was administered immediately after thetreatment described under A.

EXAMPLE 2 RELATING TO POULTRY

A and B in Example 1, with the exception of the Lactobacillusacidophilus. The preparation was administered in drinking water.

Stages A and B can be repeated as necessary at intervals.

Some of the mechanisms binding micro-organisms to phase boundaries orinterfaces are responsible for irreversible binding which acts overshort distances (2-5 nm). This results in strong adhesion which is onlymechanically broken by tearing away the components belonging to theadhered micro-organisms. Hydrophobic interaction is primarilyresponsible for this type of irreversible binding.

Successful treatment, i.e. removal of micro-organisms adhered in thismanner, is effected by modifying the quantity of substances, salts orother components, able to alter the water-structure binding propertiesin the surfaces, phase boundaries and interfaces of the system.

Experiments with infectious bacteria in wounds and micro-organismsadhered to teeth showed surprisingly that the principles and substancesused in the experiments with livestock described above were alsoextremely effective in the following widely differing fields ofapplication.

The following points constitute the basis of the process used in thesenew fields of application.

1. Substances altering the degree of hydrophobic interaction betweensurface and micro-organisms are added to the surface/phase boundariesbeing treated, so that smaller or larger portions of the irreversiblyadhere micro-organisms become detached.

2. Substances promoting adhesion of specifically suppliedmicro-organisms are added to the system treated under point 1.

The procedure is further illustrated with reference to the followingnon-limiting examples.

EXAMPLE 3

In both model system and during clinical testing using dentifrice ormouthwash with the addition of magnesium sulphate (MgSO₄) for brushingteeth or rinsing out the mouth, a significant reduction in the number ofbacteria adhered was established.

EXAMPLE 4

Bathing liquids to which magnesium sulphate and/or anti-oxidants hadbeen added were applied to a wound. Here too a significant reduction inthe number of bacteria adhered to the wound surface was noted. It wasalso noted that cleaning the surface of a wound in this mannerstimulated healing.

The result of the method is also illustrated by but not limited to thefollowing experiments in a model system.

A number of glass tubes, all the same size, were immersed in asuspension of bacteria marked with a radioactive isotope (tritium) andthe bacteria were allowed to adhere over a period of 30 minutes. Theglass tubes were then removed one by one and rinsed by repeated dipping(30 times) in a buffer solution in order to remove reversibly boundbacteria. Only the irreversibly bound bacteria then remained on theglass tube. The glass tube was then immersed in a salt solution for 5minutes. After rinsing, the quantity of bacteria adhered to the tube wasdetermined by measuring the radiation in a liquid scintillator.

A comparison of the various glass tubes which had been immersed in saltsolution of various concentrations with control tubes which hadundergone the same treatment with the exception of immersion in the saltsolution, enabled assessment of the effect of the salt solution on theirreversible bound bacteria. The table below and the drawing show theeffects of the various salts on detachment of the irreversibly adheredbacteria, expressed in percentage of the quantity of irreversiblyadhered bacteria before the salt treatment. The concentrations of thesolutions are in multiples of a specific critical concentration (C) ofthe basic solution.

Of the salts tested in this series of experiments, magnesium sulphate(MgSO₄) gave the strongest detaching effect. However, magnesium chloride(MgCl) and ammonium acetate (NH₄ Ac) solutions also gave good results,whereas ammonium sulphate ((NH₄)₂ SO₄) solutions gave a slightlydifferent pattern.

Several other compounds and salts have been tested besides thosementioned in this series of experiments, and the results were similar.

                  TABLE                                                           ______________________________________                                        Detachment percentage of irreversibly adhered bacteria after                  treatment.                                                                    The number of bacteria irreversibly adhered to the glass tube is              3.0 × 10.sup.6 before treatment.                                               Salt concentration in multiples of basic solution C                    Salts    0.05 × C                                                                          0.1 × C                                                                           0.2 × C                                                                         0.6 × C                            ______________________________________                                        MgSO.sub.4                                                                             41        54        28      28                                       MgCl.sub.2                                                                             20        40        21      25                                       NH.sub.4 Acetate                                                                       8.5       41        17      24                                       (NH.sub.4)SO.sub.4                                                                     31        36        44      28                                       ______________________________________                                    

FIG. 1 shows the detachment of irreversibly adhered bacteria aftertreatment with salt solutions of various concentrations. The saltconcentration is expressed in multiples of the basic solution C. Thenumber of irreversibly adhered bacteria is ca. 3.0×10⁶ per glass tubebefore treatment.

The preparations containing curcumine described in the following claimsare intended for use in performing the method according to theinvention, but can also be used for other purposes, such as stimulatingsecretion in micro-organisms tissues and tissue cells.

We claim:
 1. Method for removing undesired bacteria and promoting theestablishment of desired bacteria on an interfacial surface, comprisingthe steps of:(a) treating said surface with an effective amount ofmaterial which will substantially remove the bacteria attached to saidsurface; (b) applying to said treated surface an effective amount of anadhesion-promoting material comprising curcumin or a derivative thereof;and (c) applying to said treated surface a desired, fimbriated bacteria.2. Method according to claim 1, wherein said surface is treated with anorganic and/or inorganic salt for substantial removal of the attachedbacteria.
 3. Method according to claim 2, wherein said said salt isselected from the group consisting of salts of magnesium, aluminum,ammonium, beryllium, lithium, calcium, and mixtures thereof.
 4. Methodaccording to claim 1, wherein said surface is treated with erythrosineand/or an anti-oxidant for substantial removal of the attached bacteria.5. Method according to claim 2, wherein said surface is treated witherythrosine and/or an anti-oxidant together with said salt forsubstantial removal of the attached bacteria.
 6. Method according toclaim 1, wherein said adhesion-promoting material is a derivative ofcurcumin of the structural formula: ##STR3## wherein n is 1, 2 or 3, xis 1, 2, 3 or 4, and R₁ -R₅ are independently selected from the groupconsisting of H, OH, OCH₃, OCH₂ CH₃, CH₃, CH₂ CH₃, COOH, and CH₂ -COOH.7. Method according to claim 1, wherein said curcumin is applied in theform oleo-resins and/or cleaned fractions of a plant extract containingcurcumin, which also includes turmeric acid.
 8. Method according toclaim 1, wherein said surface is a surface of the mouth, stomach, orintestine of a living animal.
 9. Method according to claim 8, whereinsaid step of treating is by oral administration.
 10. Method according toclaim 8, wherein said steps of applying are by oral administration. 11.Method according to claim 10, wherein curcumin is orally administered inan amount of 0.8 g/kg body weight of said living animal.
 12. Compositionfor performing the method according to claim 1, comprising an organicand/or an inorganic salt selected from the group consisting of lithium,beryllium, magnesium, calcium, aluminum, and ammonium salts, andmixtures thereof, to remove bacteria.
 13. Composition for performing themethod according to claim 1, comprising erythrosine and/or anti-oxidantsto remove bacteria.
 14. Composition for performing the method accordingto claim 1, comprising curcumin or a derivative thereof as an adhesionstimulating substance.
 15. Composition according to claim 10, comprisingcurcumin in the form of oleo-resins or plant extracts including curcuminand turmenic acid.
 16. Composition for performing the method accordingto claim 1, in the form of an ointment, cream or bathing liquid. 17.Composition for performing the method according to claim 1 in the formof a dentifrice comprising at least one organic and/or inorganic salt oflithium, beryllium, magnesium, calcium, ammonium, or aluminum.
 18. Aprocess for promoting the adhesion of a desired bacteria to aninterfacial surface, comprising applying to said surface an effectiveamount of an adhesion promoting substance comprising curcumin or aderivative thereof, and applying to said surface fimbriated desiredbacteria.